CN113563480B - 一种cld蛋白突变体及应用 - Google Patents
一种cld蛋白突变体及应用 Download PDFInfo
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- CN113563480B CN113563480B CN202110786982.1A CN202110786982A CN113563480B CN 113563480 B CN113563480 B CN 113563480B CN 202110786982 A CN202110786982 A CN 202110786982A CN 113563480 B CN113563480 B CN 113563480B
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- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
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Abstract
本发明涉及基因工程领域，更具体涉及一种CLD蛋白突变体及应用。所述的蛋白为SEQ ID NO.1‑SEQ ID NO.5所示的任一一个蛋白，申请人将原代CLD重组蛋白中的CD4 60位处的半胱氨酸突变为丝氨酸，获得的CLD蛋白突变体对测试的HIV‑1毒株抑制能力提高2‑3个数量级，显著提高了对病毒的中和能力，这种差异在测试的HIV‑1 T/F(transmitter/founder)virus中更加显著，CLD蛋白能够大幅提高与HIV‑1病毒的结合效率；CLD蛋白突变体或重组CLD蛋白与包膜蛋白形成的复合物可以作为一种免疫原，更好诱导出针对包膜蛋白V1V2区的抗体，表现出良好的应用前景。
Description
技术领域
本发明涉及基因工程领域，更具体涉及一种CLD蛋白突变体及应用。
背景技术
人类免疫缺陷病毒(HIV-1)是艾滋病(AIDS，获得性免疫缺陷综合征)的病原。由于HIV-1的高度变异及对人类免疫的机制了解的缺乏，目前尚未开发出成功的HIV-1疫苗。对HIV-1感染者的治疗及预防，主要依赖于抗HIV药物。但由于HIV-1高度的变异性，临床药物长期使用必然导致病毒抗、耐药性。开发新型抗病毒药物对HIV-1治疗的可持续性具有迫切性。
HIV-1感染的靶细胞类型包括T细胞、巨噬细胞及部分类型的DC细胞，其共同特征是细胞表面表达CD4分子及辅助受体分子。根据HIV-1在感染细胞过程中使用的是CCR5还是CXCR4辅助受体可将HIV-1分为R5和X4病毒，有些亚型艾滋病毒还存在使用CCR5和CXCR4的中间类型R5X4病毒。HIV-1感染靶标细胞的过程实是病毒包膜蛋白识别、结合CD4和辅助受体的过程。但无论是R5，X4还是R5X4病毒都要利用CD4才能完成感染过程。HIV-1包膜蛋白与CD4及辅助受体的结合足以使病毒感染细胞。因此，以HIV-1包膜蛋白CD4结合位点为靶标可以阻断HIV-1感染细胞。
DC-SIGN是一种表达在DC细胞表面的凝集素识别蛋白，可以通过结合包膜蛋白表面的多糖富集病毒，可溶性DC-SIGN可以抑制HIV-1包膜蛋白与DC细胞的结合。
早期申请人尝试将CD4与DC-SIGN融合后在原核细胞内表达(CN 102617738A)，结果显示设计的重组蛋白CLD具有较好的抗病毒活性，测试的病毒中和能力达到微克水平，但申请人在后期的实验中发现，该重组蛋白对大部分T/F毒株效果不佳。
针对上述问题，本申请对融合重组蛋白进行了改进，将重组融合蛋白CD4结构域的60位的半胱氨酸突变为丝氨酸，去掉了原核表达中使用的HIS组氨酸序列，在真核系统中进行了表达，所获得的重组蛋白CLD突变体相对原核表达的第一代CLD活性得到了极大的提高，也具有很强的广谱性，极有希望成为新一代的抗HIV-1药物。
发明内容
本发明的目的在于提供了一种CLD蛋白突变体，所述的突变体为SEQ ID NO.1～SEQ ID NO.5中的任一一个蛋白。
本发明的另一个目的在于提供了一种CLD蛋白突变体组合物，所述的组合物为SEQID NO.1～SEQ ID NO.5中的任两个、三个、四个或全部蛋白的组合。
本发明的另一个目的在于提供了CLD蛋白突变体或其组合物在制备抗HIV-1药物中的应用。
本发明的还有一个目的在于提供了一种重组CLD蛋白与HIV-1包膜蛋白组成的复合免疫原，所述的重组CLD蛋白为SEQ ID NO.1～SEQ ID NO.5中的任一一个蛋白或CN10261773 8A中的权利要求1～8中的任意一个蛋白。
本发明的最后一个目的在于提供了复合免疫原在制备抗HIV-1药物中的应用。
为了达到上述目的，本发明采取以下技术措施：
一种CLD蛋白突变体，所述的突变体为SEQ ID NO.1～SEQ ID NO.5中的任一一个蛋白，与CN 102617738A中提到的原代CLD相比，申请人将CD4 60位处的半胱氨酸突变为丝氨酸。编码产物能够大幅提高与HIV-1病毒的结合效率，并且保持蛋白的稳定性。
一种CLD蛋白突变体组合物，所述的组合物为SEQ ID NO.1～SEQ ID NO.5中的任两个、三个、四个或全部蛋白的组合。
CLD蛋白突变体在制备抗HIV-1药物中的应用，是利用SEQ ID NO.1～SEQ ID NO.5中的任一一个蛋白，或其任何组合以唯一主效成分，或是主效成分之一，用以制备抗HIV-1的药物。
一种重组CLD蛋白与HIV-1包膜蛋白组成的复合免疫原，所述的重组CLD蛋白为SEQID NO.1～SEQ ID NO.5中的任一一个蛋白或CN 102617738A中的权利要求1～8中的任意一个蛋白。
所述的HIV-1包膜蛋白包括但不限于：HIV-1 gp160、HIV-1 gp140或HIV-1gp120。
本发明的保护范围还包括，上述复合免疫原在制备抗HIV-1药物中的应用。
本发明与现有技术相比，具有以下优点和效果：
与原核重组CLD相比，真核表达的重组蛋白CLD突变体对测试的HIV-1毒株抑制能力提高2-3个数量级，显著提高了对病毒的中和能力，这种差异在测试的HIV-1T/F(transmitter/founder)virus中更加显著，表现了良好的应用前景。相比原核表达的CLD及真核表达的CLD非突变体重组融合蛋白，真核表达的CLD突变体在溶液中更加稳定，抑制HIV-1的能力变化小。在抗HIV-1感染实验中，真核表达的CLD突变体重组融合蛋白对部分毒株的抑制活性相对于于非突变体提高3-5倍。
本发明所得的系列CLD突变蛋白对HIV具有良好的抑制作用。CLD突变体蛋白在溶液状态下，形成双功能能结构域的四聚体形式，多聚化CLD突变体中的DC-SIGN功能结构域与包膜蛋白结合后，增加了四聚体化的CD4分子与包膜蛋白上CD4结合位点的局部浓度，从而提高对HIV-1中和能力。
本发明所得的一列CLD突变蛋白与HIV-1包膜蛋白组成的复合物，与CD4或DC-SIGN混合的HIV-1包膜蛋白或单独的包膜蛋白相比，作为免疫原可以诱导机体产生更强的靶向gp120 V1V2表位的免疫反应，而产生的靶向gp120 V3C3表位的免疫反应则更弱。
具体实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规方案；所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：
真核表达载体pCDNA-C25NDC60S、pCDNA-C30NDC60S、pCDNA-C35NDC60S、pCDNA-C40NDC60S和pCDNA-C45NDC60S的构建
本实施例所用到的引物如下：
P1-F：GAATTCCCTGCTGCTGCTCCTGCCTCAGGCCCAGGCTGTGAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACCTGTA；
P1-R：TTAAACGGGCCCTCTAGACTCGAGCTACGCAGGAGGGGGGTTTGGGGTG。
P2-F:ATGGACCGGGCCAAGCTGCTGCTCCTGCTCCTGCTGCTGCTCCTGCCTCTGCAGATATCCAGCACAGTGG；
P2-R:GAGGCAGGAGCAGCAGCAGGAGCAGGAGCAGCAGCTTGGCCCGGTCCATGAATTCCACCACACTGGACTAGTGG。
P3-F:GATCGCGCTGACTCAAGAAGAAGCCTTTGGGAC；
P3-R:GTCCCAAAGGCTTCTTCTTGAGTCAGCGCGATC。
(1)pCDNA-C25NDC60S的构建：
以pET28a-C25D(CN 102617738A)为模板用引物P1-F和P1-R进行PCR，核酸电泳后胶回收。以pCDNA3.1为模板用引物P2-F和P2-R进行PCR，核酸电泳后胶回收。用诺唯赞公司的同源重组试剂盒进行同源重组。取15μl重组体系加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功的命名为pCDNA-C25ND。
以构建好的pCDNA-C25ND为模板，P3-F、P3-R为引物对，使用Takara环状PCR试剂盒进行PCR扩增。每50μl PCR体系加入2μl的dpnI于37℃消化2小时，取15μl加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功则被命名为pCDNA-C25NDC60S。pCDNA-C25NDC60S编码包含CD4D1D2 N端178 aa部分和DC-SIGNNECK以及CRD部分；25个氨基酸的linker；CD460位氨基酸由半胱氨酸突变为丝氨酸
(2)pCDNA-C30NDC60S的构建：
以pET28a-C30D(CN 102617738A)为模板用引物P1-F和P1-R进行PCR，核酸电泳后胶回收。以pCDNA3.1为模板用引物P2-F和P2-R进行PCR，核酸电泳后胶回收。用诺唯赞公司的同源重组试剂盒进行同源重组。取15μl重组体系加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功的命名为pCDNA-C30ND。
以构建好的pCDNA-C30ND为模板，P3-F、P3-R为引物对，使用Takara环状PCR试剂盒进行PCR扩增。每50μl PCR体系加入2μl的dpnI于37℃消化2小时，取15μl加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功则被命名为pCDNA-C30NDC60S。pCDNA-C30NDC60S编码包含CD4D1D2 N端178 aa部分和DC-SIGNNECK以及CRD部分；30个氨基酸的linker；CD4 60位氨基酸由半胱氨酸突变为丝氨酸
(3)pCDNA-C35NDC60S的构建：
以pET28a-C35D(CN 102617738A)为模板用引物P1-F和P1-R进行PCR，核酸电泳后胶回收。以pCDNA3.1为模板用引物P2-F和P2-R进行PCR，核酸电泳后胶回收。用诺唯赞公司的同源重组试剂盒进行同源重组。取15μl重组体系加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功的命名为pCDNA-C35ND。
以构建好的pCDNA-C35ND为模板，P3-F、P3-R为引物对，使用Takara环状PCR试剂盒进行PCR扩增。每50μl PCR体系加入2μl的dpnI于37℃消化2小时，取15μl加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功则被命名为pCDNA-C35NDC60S。pCDNA-C35NDC60S编码包含CD4 D1D2 N端178 aa部分和DC-SIGNNECK以及CRD部分；35个氨基酸的linker；CD460位氨基酸由半胱氨酸突变为丝氨酸。
(4)pCDNA-C40NDC60S的构建：
以pET28a-C40D(CN 102617738A)为模板用引物P1-F和P1-R进行PCR，核酸电泳后胶回收。以pCDNA3.1为模板用引物P2-F和P2-R进行PCR，核酸电泳后胶回收。用诺唯赞公司的同源重组试剂盒进行同源重组。取15μl重组体系加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功的命名为pCDNA-C40ND。
以构建好的pCDNA-C40ND为模板，P3-F、P3-R为引物对，使用Takara环状PCR试剂盒进行PCR扩增。每50μl PCR体系加入2μl的dpnI于37℃消化2小时，取15μl加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功则被命名为pCDNA-C40NDC60S。pCDNA-C40NDC60S编码包含CD4 D1D2 N端178 aa部分和DC-SIGNNECK以及CRD部分；40个氨基酸的linker；CD460位氨基酸由半胱氨酸突变为丝氨酸。
(5)pCDNA-C45NDC60S的构建：
以pET28a-C45D为模板用引物P1-F和P1-R进行PCR，核酸电泳后胶回收。以pCDNA3.1为模板用引物P2-F和P2-R进行PCR，核酸电泳后胶回收。用诺唯赞公司的同源重组试剂盒进行同源重组。取15μl重组体系加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功的命名为pCDNA-C45ND。
以构建好的pCDNA-C45ND为模板，P3-F、P3-R为引物对，使用Takara环状PCR试剂盒进行PCR扩增。每50μl PCR体系加入2μl的dpnI于37℃消化2小时，取15μl加入到100μl大肠杆菌DH5α感受态，混匀，42℃热激转化，加入800μl LB培养液，37℃振荡培养1h，将菌液涂布在卡那霉素抗性的LB培养基上，37℃培养过夜，从转化的平板上挑取6个菌落接种至5ml含卡那霉素的LB，37℃振荡培养过夜，用质粒提取试剂盒提取质粒并测序验证，构建成功则被命名为pCDNA-C45NDC60S。pCDNA-C45NDC60S编码包含CD4 D1D2 N端178 aa部分和DC-SIGNNECK以及CRD部分；45个氨基酸的linker；CD4 60位氨基酸由半胱氨酸突变为丝氨酸。
实施例2：
实施例1制备的各真核表达载体的重组蛋白CLD突变体表达：
1.细胞培养
以60～70万/ml的细胞密度进行传代，总体积30ml，当293F细胞密度达到1.2～1.5百万个细胞/ml，收集细胞(1200转离心5min)，并用15ml培养基重悬用于转染。
2.转染：
每一百万个细胞的转染所用质粒为1～1.5μg；750μl生理盐水+37.5μg质粒；750μl生理盐水+150μl PEI(1mg/ml)。分别混匀后静置5min后，混合并温和的混匀，室温静置10min(低于20min)，然后将质粒-脂质体混合液加入摇瓶中，混匀后放入摇床(8％二氧化碳，37℃，125rpm)。4～6h后补加15ml培养基。4天后收集细胞上清，然后用50KD超滤管超滤浓缩，最终浓缩约80倍，加入10％甘油，分装存于-80℃备用。
在本发明中真核表达质粒pCDNA-C25NDC60S表达的蛋白称为C25NDC60S(SEQ IDNO.1所示)，pCDNA-C30NDC60S表达的蛋白称为C30NDC60S(SEQ ID NO.2所示)，pCDNA-C35NDC60S表达的蛋白称为C35NDC60S(SEQ ID NO.3所示)，pCDNA-C40NDC60S表达的蛋白称为C40NDC60S(SEQ ID NO.4所示)，pCDNA-C45NDC60S表达的蛋白称为C45NDC60S(SEQ IDNO.5所示)。
3.ELISA检测重组蛋白CLD突变体的浓度
(1)兔源抗DC-SIGN单克隆抗体以5μg/ml包被96孔板，50μl/孔，室温过夜；
洗板机洗脱5次，加入含1％BSA的PBS封闭液封，200μl/孔，于37℃封闭一小时；
(2)洗板机洗脱5次，孵育标准品(原核表达的CLD)/重组蛋白样品，50μl/孔，37℃孵育一小时，标准品用封闭液稀释；
(3)洗板机洗脱5次，加入课题组免疫小鼠所得的抗CLD血清，用封闭液1：1000稀释，50μl/孔，37℃孵育一小时；
(4)洗板机洗脱5次，加入HRP标记的羊抗鼠IgG的二抗，用封闭液1：10000稀释，50μl/孔，37℃孵育一小时；
(5)洗板机洗脱5次，加入提前放置室温的TMB底物，50μl/孔，避光室温孵育5分钟；(6)终止反应：加入2M H2SO4溶液，50μl/孔，使用酶标仪进行读数；
(7)绘制标准曲线，计算重组蛋白CLD突变体浓度。
利用上述方法制备的重组蛋白CLD突变体浓度为100μg/ml。
实施例3：
CLD重组蛋白及重组蛋白CLD突变体在制备治疗或预防HIV-1病毒的药物中的应用：
1)HIV-1假病毒的制备：
含有不同HIV-1env基因的pCDNA3.1(+)质粒(Centralized Facility for AIDSReag ents)与缺失HIV-1env基因的pSG3(Centralized Facility for AIDS Reagents)框架质粒经脂质体(LipofectamineTM 2000，Invitrogen Corporation)共转染293T细胞。转染48h后，含病毒的培养基上清用0.45μm滤膜过滤后加入10％体积的胎牛血清，分装到1.5ml离心管并于-80℃保存备；采用luciferase(市售，promega公司)测定病毒滴度。
上述不同的的pCDNA3.1(+)质粒含有的不同HIV-1env基因为：MSW2、CH811、700010040.C9.4520、PRB958_06.TB1.4305、WEAUd15.410.787、62357_14.D3.4589、REJO.D12.1972、SC05.8C11.2344、1059_09.A4.1460、6240_08.TA5.4622、700010058.A4.4375、1058_11.B11.1550、SC45.4B5.2631、62615_03.P4.3964。
2)HIV-1真病毒的制备：
包含HIV-1全基因组的不同质粒经由脂质体(LipofectamineTM 2000，InvitrogenCorpor ation)转染293T细胞。转染48h后，含病毒的培养基上清用0.45μm滤膜过滤后加入10％体积的胎牛血清，分装到1.5ml离心管并保存于-80℃保存备；采用luciferase(市售，promega公司)测定病毒滴度。
上述不同质粒为实验室适应株NL4-3和BaL；T/F株包含THRO.c/2626、CH077.t/2627、CH040.c/2625、pCH058.c/2960、WITO.c/2474、SUMA.c/2821、CH164、CH185、CH198。
3)各重组蛋白抑制HIV-1感染TZM-bl细胞系：
A.将各重组蛋白溶液稀释至1μM，并以此为起始浓度，以3为稀释系数向下稀释11个梯度，最后加上一个不含重组蛋白的培养基作为对照；
B.将病毒稀释到200TCID50；
C.60μl稀释好的病毒与60μl各重组蛋白的稀释液混合，37℃孵育1小时；
D.取100μl病毒-重组蛋白混合液加入到提前铺至96孔板的TZM-bl细胞中，随后加入100μl含DEAE(40μg/ml)的完全培养基，于二氧化碳培养箱中培养48小时；
E.采用luciferase(市售，promega公司)测定荧光值；
F.计算抑制率，以不加CLD孔的读数作为0％抑制率。
结果如下表所示：
C25ND和C35ND为CN 102617738A中报道的原核表达的CLD重组蛋白：
所述的C35NDS60C蛋白来源于《Bifunctional CD4–DC-SIGN Fusion ProteinsDemonst rate Enhanced Avidity to gp120 and Inhibit HIV-1 Infection andDissemination》。
上述表格的空白表明并未做该项数据。
结果表明CLD蛋白突变体与对各类型HIV-1病毒抑制效果均比CN 102617738A中报道的原核表达的CLD重组蛋白要好。
实施例4：
CN 102617738A申请中的8个重组蛋白CLD任一一个与HIV-1包膜蛋白制备混合免疫原在制备预防HIV-1病毒药物中的应用：
1)重组蛋白CLD、CD4和DC-SIGN与HIV-1包膜蛋白混合免疫原的制备：
实验组：重组蛋白CLD与HIV-1包膜蛋白(5μg)按照3：1(摩尔比)的比例混合，并于4℃孵育24小时，一共100μl；
所述的重组蛋白CLD为CN 102617738A申请中权利要求1-8中的任一蛋白。
对照组1：CD4与HIV-1包膜蛋白(5μg)按照12：1(摩尔比)的比例进行混合的混合物，并于4℃孵育24小时，一共100μl；
对照组2：DC-SIGN与HIV-1包膜蛋白(5μg)按照12：1(摩尔比)的比例进行混合的混合物，并于4℃孵育24小时，一共100μl；
重组蛋白CLD是以四聚体形式存在的，一个CLD四聚体含有四个CD4和四个DC-SIGN，所以对照使用12：1的摩尔比。
所述的HIV-1包膜蛋白为HIV-1 CN54的gp140蛋白。
2)免疫小鼠：皮下注射100μl步骤1)的各组别试剂，共免疫3次，每两次免疫之间间隔3周，最后一次免疫后的第7天处死小鼠，并收集血清和脾脏；
3)实验方法
A.为了探究CD4结合gp140能否影响其构象改变并暴露CD4i表位，本次实验我们选用了17b(CD4i)、19b(CD4i)、447-52D(V3)、39F(V3)、12b(CD4BS)和F105 6个识别gp120不同靶位的单克隆抗体进行ELISA实验。用gp140或者gp140与重组蛋白CLD混合物包被96孔板(0.25μg/孔)在室温下过夜，TBST洗涤三次，接着用含1％BSA的TBST在37℃封闭1h。连续梯度稀释的上述抗体在37℃孵育1h。TBST三次洗涤后用HRP标记的山羊抗小鼠二抗(1:5000稀释)在37℃孵育1h。经过5次洗涤后,加入TMB室温避光孵育5min，然后加入2M浓硫酸终止反应。最后用酶标仪检测OD值，以450nm为实验波长，以570nm为参比波长。
B.为了探究ELISA检测血清中gp140特异性抗体滴度。用gp140或gp140与sCD4混合物包被96孔板(0.25μg/孔)在室温下过夜，TBST洗涤三次，接着用含1％BSA的TBST在37℃封闭1h。连续梯度稀释的样本在37C孵育1h。TBST三次洗涤后，用,HRP标记的山羊抗小鼠二抗(1:5000稀释)在37℃孵育1h。经过5次洗涤后,加入TMB室温避光孵育5min，然后加入2M浓硫酸终止反应。最后用酶标仪检测OD值，以450nm为实验波长，以570nm为参比波长。
C.细胞因子检测
取实验组小鼠脾脏，分离淋巴细胞。以每孔3×107细胞数铺到24孔板中，用gp140(20μg/孔)或者CLD-gp140(35μg/孔)刺激，5天后收上清，用0.22um滤器过滤，分装后存-80℃备用。用BD Biosciences细胞因子试剂盒检测上清中IL-2,IL-4,IL-5,IFN-γ和TNF-α的量。
4)实验结果
CN 102617738A申请中重组蛋白CLD影响mAbs(17b,19b,447-52D,39F,b12,F105)结合HIV-1 gp140。
CN 102617738A申请中重组蛋白CLD与HIV-1包膜蛋白组成的复合物，与CD4或DC-SIGN混合的HIV-1包膜蛋白或单独的包膜蛋白相比，作为免疫原可以诱导机体产生更强的靶向gp120 V1V2表位的抗体反应，而产生的靶向gp120 V3C3表位的抗体反应则更弱。
CN 102617738A申请中重组蛋白CLD与HIV-1包膜蛋白组成的复合物，与CD4或DC-SIGN混合的HIV-1包膜蛋白或单独的包膜蛋白相比，作为免疫原可以诱导机体产生不同的gp140特异性的Th1/Th2细胞免疫反应。CLD突变体与HIV-1包膜蛋白组成的复合物免疫的小鼠脾脏细胞中，gp140特异性的表达IL-4、IL-5和IFN-γ的细胞显著降低；表达TNF的细胞也有降低，但是没有显著性差异。
实施例5：
CLD蛋白突变体与HIV-1包膜蛋白混合免疫原在预防HIV-1病毒中的应用：
1)CLD蛋白突变体、CD4和DC-SIGN与HIV-1包膜蛋白混合免疫原的制备：
实验组：CLD蛋白突变体与HIV-1包膜蛋白(5μg)按照3：1(摩尔比)的比例混合，并于4℃孵育24小时，一共100μl；
所述的CLD蛋白突变体为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4或SEQ ID NO.5所示蛋白。
对照组1：CD4与HIV-1包膜蛋白(5μg)按照12：1(摩尔比)的比例进行混合的混合物，并于4℃孵育24小时，一共100μl；
对照组2：DC-SIGN与HIV-1包膜蛋白(5μg)按照12：1(摩尔比)的比例进行混合的混合物，并于4℃孵育24小时，一共100μl；
CLD是以四聚体形式存在的，一个CLD四聚体含有四个CD4和四个DC-SIGN，所以对照使用12：1的摩尔比。
所述的HIV-1包膜蛋白为HIV-1 CN54的gp140蛋白。
2)免疫小鼠：皮下注射100μl步骤1)的各组别试剂，共免疫3次，每两次免疫之间间隔3周，最后一次免疫后的第7天处死小鼠，并收集血清和脾脏；
3)实验方法
A.为了探究CD4结合gp140能否影响其构象改变并暴露CD4i表位，本次实验我们选用了17b(CD4i)、19b(CD4i)、447-52D(V3)、39F(V3)、12b(CD4BS)和F105等6个识别gp120不同靶位的单克隆抗体进行ELISA实验。用gp140或者gp140与重组蛋白CLD混合物包被96孔板(0.25μg/孔)在室温下过夜，TBST洗涤三次，接着用含1％BSA的TBST在37℃封闭1h。连续梯度稀释的上述抗体在37℃孵育1h。TBST三次洗涤后用HRP标记的山羊抗小鼠二抗(1:5000稀释)在37℃孵育1h。经过5次洗涤后,加入TMB室温避光孵育5min，然后加入2M浓硫酸终止反应。最后用酶标仪检测OD值，以450nm为实验波长，以570nm为参比波长。
B.为了探究ELISA检测血清中gp140特异性抗体滴度。用gp140或gp140与sCD4混合物包被96孔板(0.25μg/孔)在室温下过夜,TBST洗涤三次，接着用含1％BSA的TBST在37℃封闭1h。连续梯度稀释的样本在37C孵育1h。TBST三次洗涤后，用,HRP标记的山羊抗小鼠二抗(1:5000稀释)在37℃孵育1h。经过5次洗涤后,加入TMB室温避光孵育5min，然后加入2M浓硫酸终止反应。最后用酶标仪检测OD值，以450nm为实验波长，以570nm为参比波长。
C.细胞因子检测
取实验组小鼠脾脏，分离淋巴细胞。以每孔3×107细胞数铺到24孔板中，用gp140(20μg/孔)或者CLD-gp140(35μg/孔)刺激，5天后收上清，用0.22um滤器过滤，分装后存-80℃备用。用BD Biosciences细胞因子试剂盒检测上清中IL-2,IL-4,IL-5,IFN-γ和TNF-α的量。
4)实验结果
相比实施例4中重组蛋白CLD，本发明CLD突变体影响mAbs(17b,19b,447-52D,39F,b12,F105)结合HIV-1 gp140更强。
本发明CLD蛋白突变体与HIV-1包膜蛋白组成的复合物，与CD4或DC-SIGN混合的HIV-1包膜蛋白或单独的包膜蛋白相比，作为免疫原可以诱导机体产生更强的靶向gp120V1V2表位的抗体反应，而产生的靶向gp120 V3C3表位的抗体反应则更弱。而且相较于实施例4中重组蛋白CLD，产生的差异更明显。
本发明CLD突变体与HIV-1包膜蛋白组成的复合物，与CD4或DC-SIGN混合的HIV-1包膜蛋白或单独的包膜蛋白相比，作为免疫原可以诱导机体产生不同的gp140特异性的Th1/Th2细胞免疫反应。CLD突变体与HIV-1包膜蛋白组成的复合物免疫的小鼠脾脏细胞中，gp140特异性的表达IL-4、IL-5、TNF和IFN-γ的细胞显著降低。而且相较于实施例4中重组蛋白CLD，产生的差异更明显。
序列表
<110> 成都维瑾柏鳌生物医药科技有限公司
<120> 一种CLD蛋白突变体及应用
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 541
<212> PRT
<213>人工序列
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Met Asp Arg Ala Lys Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro
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Gln Ala Gln Ala Val Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr
20 25 30
Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His
35 40 45
Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe
50 55 60
Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg
65 70 75 80
Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys
85 90 95
Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu
100 105 110
Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His
115 120 125
Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly
130 135 140
Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln
145 150 155 160
Gly Glu Lys Thr Leu Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly
165 170 175
Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys
180 185 190
Ile Asp Ile Val Val Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
195 200 205
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Val Gly Glu Leu Ser Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu
225 230 235 240
Leu Thr Gln Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys
245 250 255
Leu Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly
260 265 270
Glu Leu Pro Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr
275 280 285
Trp Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Met Gln
290 295 300
Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu
305 310 315 320
Pro Glu Lys Ser Lys Gln Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu
325 330 335
Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gln Gln Glu Ile
340 345 350
Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu
355 360 365
Lys Ser Lys Gln Gln Glu Ile Tyr Gln Glu Leu Thr Gln Leu Lys Ala
370 375 380
Ala Val Glu Arg Leu Cys His Pro Cys Pro Trp Glu Trp Thr Phe Phe
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Gln Gly Asn Cys Tyr Phe Met Ser Asn Ser Gln Arg Asn Trp His Asp
405 410 415
Ser Ile Thr Ala Cys Lys Glu Val Gly Ala Gln Leu Val Val Ile Lys
420 425 430
Ser Ala Glu Glu Gln Asn Phe Leu Gln Leu Gln Ser Ser Arg Ser Asn
435 440 445
Arg Phe Thr Trp Met Gly Leu Ser Asp Leu Asn Gln Glu Gly Thr Trp
450 455 460
Gln Trp Val Asp Gly Ser Pro Leu Leu Pro Ser Phe Lys Gln Tyr Trp
465 470 475 480
Asn Arg Gly Glu Pro Asn Asn Val Gly Glu Glu Asp Cys Ala Glu Phe
485 490 495
Ser Gly Asn Gly Trp Asn Asp Asp Lys Cys Asn Leu Ala Lys Phe Trp
500 505 510
Ile Cys Lys Lys Ser Ala Ala Ser Cys Ser Arg Asp Glu Glu Gln Phe
515 520 525
Leu Ser Pro Ala Pro Ala Thr Pro Asn Pro Pro Pro Ala
530 535 540
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<212> PRT
<213> 人工序列
<400> 2
Met Asp Arg Ala Lys Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro
1 5 10 15
Gln Ala Gln Ala Val Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr
20 25 30
Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His
35 40 45
Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe
50 55 60
Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg
65 70 75 80
Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys
85 90 95
Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu
100 105 110
Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His
115 120 125
Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly
130 135 140
Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln
145 150 155 160
Gly Glu Lys Thr Leu Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly
165 170 175
Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys
180 185 190
Ile Asp Ile Val Val Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
195 200 205
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Val Gly Glu Leu Ser Glu Lys Ser Lys Leu Gln
225 230 235 240
Glu Ile Tyr Gln Glu Leu Thr Gln Leu Lys Ala Ala Val Gly Glu Leu
245 250 255
Pro Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu
260 265 270
Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Leu Gln Glu Ile
275 280 285
Tyr Gln Glu Leu Thr Trp Leu Lys Ala Ala Val Gly Glu Leu Pro Glu
290 295 300
Lys Ser Lys Met Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala
305 310 315 320
Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gln Gln Glu Ile Tyr Gln
325 330 335
Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser
340 345 350
Lys Gln Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val
355 360 365
Gly Glu Leu Pro Glu Lys Ser Lys Gln Gln Glu Ile Tyr Gln Glu Leu
370 375 380
Thr Gln Leu Lys Ala Ala Val Glu Arg Leu Cys His Pro Cys Pro Trp
385 390 395 400
Glu Trp Thr Phe Phe Gln Gly Asn Cys Tyr Phe Met Ser Asn Ser Gln
405 410 415
Arg Asn Trp His Asp Ser Ile Thr Ala Cys Lys Glu Val Gly Ala Gln
420 425 430
Leu Val Val Ile Lys Ser Ala Glu Glu Gln Asn Phe Leu Gln Leu Gln
435 440 445
Ser Ser Arg Ser Asn Arg Phe Thr Trp Met Gly Leu Ser Asp Leu Asn
450 455 460
Gln Glu Gly Thr Trp Gln Trp Val Asp Gly Ser Pro Leu Leu Pro Ser
465 470 475 480
Phe Lys Gln Tyr Trp Asn Arg Gly Glu Pro Asn Asn Val Gly Glu Glu
485 490 495
Asp Cys Ala Glu Phe Ser Gly Asn Gly Trp Asn Asp Asp Lys Cys Asn
500 505 510
Leu Ala Lys Phe Trp Ile Cys Lys Lys Ser Ala Ala Ser Cys Ser Arg
515 520 525
Asp Glu Glu Gln Phe Leu Ser Pro Ala Pro Ala Thr Pro Asn Pro Pro
530 535 540
Pro Ala
545
<210> 3
<211> 551
<212> PRT
<213> 人工序列
<400> 3
Met Asp Arg Ala Lys Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro
1 5 10 15
Gln Ala Gln Ala Val Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr
20 25 30
Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His
35 40 45
Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe
50 55 60
Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg
65 70 75 80
Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys
85 90 95
Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu
100 105 110
Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His
115 120 125
Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly
130 135 140
Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln
145 150 155 160
Gly Glu Lys Thr Leu Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly
165 170 175
Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys
180 185 190
Ile Asp Ile Val Val Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
195 200 205
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Gly Glu Leu Ser Glu
225 230 235 240
Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Gln Leu Lys Ala
245 250 255
Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln
260 265 270
Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser
275 280 285
Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Trp Leu Lys Ala Ala Val
290 295 300
Gly Glu Leu Pro Glu Lys Ser Lys Met Gln Glu Ile Tyr Gln Glu Leu
305 310 315 320
Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gln
325 330 335
Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu
340 345 350
Leu Pro Glu Lys Ser Lys Gln Gln Glu Ile Tyr Gln Glu Leu Thr Arg
355 360 365
Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gln Gln Glu
370 375 380
Ile Tyr Gln Glu Leu Thr Gln Leu Lys Ala Ala Val Glu Arg Leu Cys
385 390 395 400
His Pro Cys Pro Trp Glu Trp Thr Phe Phe Gln Gly Asn Cys Tyr Phe
405 410 415
Met Ser Asn Ser Gln Arg Asn Trp His Asp Ser Ile Thr Ala Cys Lys
420 425 430
Glu Val Gly Ala Gln Leu Val Val Ile Lys Ser Ala Glu Glu Gln Asn
435 440 445
Phe Leu Gln Leu Gln Ser Ser Arg Ser Asn Arg Phe Thr Trp Met Gly
450 455 460
Leu Ser Asp Leu Asn Gln Glu Gly Thr Trp Gln Trp Val Asp Gly Ser
465 470 475 480
Pro Leu Leu Pro Ser Phe Lys Gln Tyr Trp Asn Arg Gly Glu Pro Asn
485 490 495
Asn Val Gly Glu Glu Asp Cys Ala Glu Phe Ser Gly Asn Gly Trp Asn
500 505 510
Asp Asp Lys Cys Asn Leu Ala Lys Phe Trp Ile Cys Lys Lys Ser Ala
515 520 525
Ala Ser Cys Ser Arg Asp Glu Glu Gln Phe Leu Ser Pro Ala Pro Ala
530 535 540
Thr Pro Asn Pro Pro Pro Ala
545 550
<210> 4
<211> 556
<212> PRT
<213>人工序列
<400> 4
Met Asp Arg Ala Lys Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro
1 5 10 15
Gln Ala Gln Ala Val Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr
20 25 30
Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His
35 40 45
Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe
50 55 60
Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg
65 70 75 80
Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys
85 90 95
Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu
100 105 110
Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His
115 120 125
Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly
130 135 140
Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln
145 150 155 160
Gly Glu Lys Thr Leu Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly
165 170 175
Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys
180 185 190
Ile Asp Ile Val Val Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
195 200 205
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val
225 230 235 240
Gly Glu Leu Ser Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu
245 250 255
Thr Gln Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Leu
260 265 270
Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu
275 280 285
Leu Pro Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Trp
290 295 300
Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Met Gln Glu
305 310 315 320
Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro
325 330 335
Glu Lys Ser Lys Gln Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys
340 345 350
Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gln Gln Glu Ile Tyr
355 360 365
Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys
370 375 380
Ser Lys Gln Gln Glu Ile Tyr Gln Glu Leu Thr Gln Leu Lys Ala Ala
385 390 395 400
Val Glu Arg Leu Cys His Pro Cys Pro Trp Glu Trp Thr Phe Phe Gln
405 410 415
Gly Asn Cys Tyr Phe Met Ser Asn Ser Gln Arg Asn Trp His Asp Ser
420 425 430
Ile Thr Ala Cys Lys Glu Val Gly Ala Gln Leu Val Val Ile Lys Ser
435 440 445
Ala Glu Glu Gln Asn Phe Leu Gln Leu Gln Ser Ser Arg Ser Asn Arg
450 455 460
Phe Thr Trp Met Gly Leu Ser Asp Leu Asn Gln Glu Gly Thr Trp Gln
465 470 475 480
Trp Val Asp Gly Ser Pro Leu Leu Pro Ser Phe Lys Gln Tyr Trp Asn
485 490 495
Arg Gly Glu Pro Asn Asn Val Gly Glu Glu Asp Cys Ala Glu Phe Ser
500 505 510
Gly Asn Gly Trp Asn Asp Asp Lys Cys Asn Leu Ala Lys Phe Trp Ile
515 520 525
Cys Lys Lys Ser Ala Ala Ser Cys Ser Arg Asp Glu Glu Gln Phe Leu
530 535 540
Ser Pro Ala Pro Ala Thr Pro Asn Pro Pro Pro Ala
545 550 555
<210> 5
<211> 561
<212> PRT
<213> 人工序列
<400> 5
Met Asp Arg Ala Lys Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro
1 5 10 15
Gln Ala Gln Ala Val Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr
20 25 30
Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His
35 40 45
Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe
50 55 60
Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg
65 70 75 80
Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys
85 90 95
Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu
100 105 110
Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His
115 120 125
Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly
130 135 140
Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln
145 150 155 160
Gly Glu Lys Thr Leu Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly
165 170 175
Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys
180 185 190
Ile Asp Ile Val Val Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
195 200 205
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Val Gly Glu Leu Ser Glu Lys Ser Lys Leu Gln Glu
245 250 255
Ile Tyr Gln Glu Leu Thr Gln Leu Lys Ala Ala Val Gly Glu Leu Pro
260 265 270
Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys
275 280 285
Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Leu Gln Glu Ile Tyr
290 295 300
Gln Glu Leu Thr Trp Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys
305 310 315 320
Ser Lys Met Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala
325 330 335
Val Gly Glu Leu Pro Glu Lys Ser Lys Gln Gln Glu Ile Tyr Gln Glu
340 345 350
Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys
355 360 365
Gln Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly
370 375 380
Glu Leu Pro Glu Lys Ser Lys Gln Gln Glu Ile Tyr Gln Glu Leu Thr
385 390 395 400
Gln Leu Lys Ala Ala Val Glu Arg Leu Cys His Pro Cys Pro Trp Glu
405 410 415
Trp Thr Phe Phe Gln Gly Asn Cys Tyr Phe Met Ser Asn Ser Gln Arg
420 425 430
Asn Trp His Asp Ser Ile Thr Ala Cys Lys Glu Val Gly Ala Gln Leu
435 440 445
Val Val Ile Lys Ser Ala Glu Glu Gln Asn Phe Leu Gln Leu Gln Ser
450 455 460
Ser Arg Ser Asn Arg Phe Thr Trp Met Gly Leu Ser Asp Leu Asn Gln
465 470 475 480
Glu Gly Thr Trp Gln Trp Val Asp Gly Ser Pro Leu Leu Pro Ser Phe
485 490 495
Lys Gln Tyr Trp Asn Arg Gly Glu Pro Asn Asn Val Gly Glu Glu Asp
500 505 510
Cys Ala Glu Phe Ser Gly Asn Gly Trp Asn Asp Asp Lys Cys Asn Leu
515 520 525
Ala Lys Phe Trp Ile Cys Lys Lys Ser Ala Ala Ser Cys Ser Arg Asp
530 535 540
Glu Glu Gln Phe Leu Ser Pro Ala Pro Ala Thr Pro Asn Pro Pro Pro
545 550 555 560
Ala
<210> 6
<211> 89
<212> DNA
<213> 人工序列
<400> 6
gaattccctg ctgctgctcc tgcctcaggc ccaggctgtg aagaaagtgg tgctgggcaa 60
aaaaggggat acagtggaac tgacctgta 89
<210> 7
<211> 49
<212> DNA
<400> 7
ttaaacgggc cctctagact cgagctacgc aggagggggg tttggggtg 49
<210> 8
<211> 70
<212> DNA
<213> 人工序列
<400> 8
atggaccggg ccaagctgct gctcctgctc ctgctgctgc tcctgcctct gcagatatcc 60
agcacagtgg 70
<210> 9
<211> 74
<212> DNA
<213> 人工序列
<400> 9
gaggcaggag cagcagcagg agcaggagca gcagcttggc ccggtccatg aattccacca 60
cactggacta gtgg 74
<210> 10
<211> 33
<212> DNA
<213> 人工序列
<400> 10
gatcgcgctg actcaagaag aagcctttgg gac 33
<210> 11
<211> 33
<212> DNA
<213> 人工序列
<400> 11
gtcccaaagg cttcttcttg agtcagcgcg atc 33
<210> 12
<211> 365
<212> PRT
<213> 人工序列
<400> 12
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr
20 25 30
Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His
35 40 45
Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe
50 55 60
Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg
65 70 75 80
Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys
85 90 95
Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu
100 105 110
Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His
115 120 125
Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly
130 135 140
Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln
145 150 155 160
Gly Glu Lys Thr Leu Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly
165 170 175
Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys
180 185 190
Ile Asp Ile Val Val Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
195 200 205
Ser Gly Gly Gly Gly Ser His Pro Cys Pro Trp Glu Trp Thr Phe Phe
210 215 220
Gln Gly Asn Cys Tyr Phe Met Ser Asn Ser Gln Arg Asn Trp His Asp
225 230 235 240
Ser Ile Thr Ala Cys Lys Glu Val Gly Ala Gln Leu Val Val Ile Lys
245 250 255
Ser Ala Glu Glu Gln Asn Phe Leu Gln Leu Gln Ser Ser Arg Ser Asn
260 265 270
Arg Phe Thr Trp Met Gly Leu Ser Asp Leu Asn Gln Glu Gly Thr Trp
275 280 285
Gln Trp Val Asp Gly Ser Pro Leu Leu Pro Ser Phe Lys Gln Tyr Trp
290 295 300
Asn Arg Gly Glu Pro Asn Asn Val Gly Glu Glu Asp Cys Ala Glu Phe
305 310 315 320
Ser Gly Asn Gly Trp Asn Asp Asp Lys Cys Asn Leu Ala Lys Phe Trp
325 330 335
Ile Cys Lys Lys Ser Ala Ala Ser Cys Ser Arg Asp Glu Glu Gln Phe
340 345 350
Leu Ser Pro Ala Pro Ala Thr Pro Asn Pro Pro Pro Ala
355 360 365
<210> 13
<211> 385
<212> PRT
<213> 人工序列
<400> 13
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr
20 25 30
Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His
35 40 45
Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe
50 55 60
Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg
65 70 75 80
Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys
85 90 95
Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu
100 105 110
Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His
115 120 125
Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly
130 135 140
Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln
145 150 155 160
Gly Glu Lys Thr Leu Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly
165 170 175
Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys
180 185 190
Ile Asp Ile Val Val Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
195 200 205
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Pro Cys Pro Trp Glu
225 230 235 240
Trp Thr Phe Phe Gln Gly Asn Cys Tyr Phe Met Ser Asn Ser Gln Arg
245 250 255
Asn Trp His Asp Ser Ile Thr Ala Cys Lys Glu Val Gly Ala Gln Leu
260 265 270
Val Val Ile Lys Ser Ala Glu Glu Gln Asn Phe Leu Gln Leu Gln Ser
275 280 285
Ser Arg Ser Asn Arg Phe Thr Trp Met Gly Leu Ser Asp Leu Asn Gln
290 295 300
Glu Gly Thr Trp Gln Trp Val Asp Gly Ser Pro Leu Leu Pro Ser Phe
305 310 315 320
Lys Gln Tyr Trp Asn Arg Gly Glu Pro Asn Asn Val Gly Glu Glu Asp
325 330 335
Cys Ala Glu Phe Ser Gly Asn Gly Trp Asn Asp Asp Lys Cys Asn Leu
340 345 350
Ala Lys Phe Trp Ile Cys Lys Lys Ser Ala Ala Ser Cys Ser Arg Asp
355 360 365
Glu Glu Gln Phe Leu Ser Pro Ala Pro Ala Thr Pro Asn Pro Pro Pro
370 375 380
Ala
385
<210> 14
<211> 541
<212> PRT
<213> 人工序列
<400> 14
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr
20 25 30
Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His
35 40 45
Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe
50 55 60
Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg
65 70 75 80
Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys
85 90 95
Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu
100 105 110
Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His
115 120 125
Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly
130 135 140
Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln
145 150 155 160
Gly Glu Lys Thr Leu Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly
165 170 175
Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys
180 185 190
Ile Asp Ile Val Val Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
195 200 205
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Val Gly Glu Leu Ser Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu
225 230 235 240
Leu Thr Gln Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys
245 250 255
Leu Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly
260 265 270
Glu Leu Pro Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr
275 280 285
Trp Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Met Gln
290 295 300
Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu
305 310 315 320
Pro Glu Lys Ser Lys Gln Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu
325 330 335
Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gln Gln Glu Ile
340 345 350
Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu
355 360 365
Lys Ser Lys Gln Gln Glu Ile Tyr Gln Glu Leu Thr Gln Leu Lys Ala
370 375 380
Ala Val Glu Arg Leu Cys His Pro Cys Pro Trp Glu Trp Thr Phe Phe
385 390 395 400
Gln Gly Asn Cys Tyr Phe Met Ser Asn Ser Gln Arg Asn Trp His Asp
405 410 415
Ser Ile Thr Ala Cys Lys Glu Val Gly Ala Gln Leu Val Val Ile Lys
420 425 430
Ser Ala Glu Glu Gln Asn Phe Leu Gln Leu Gln Ser Ser Arg Ser Asn
435 440 445
Arg Phe Thr Trp Met Gly Leu Ser Asp Leu Asn Gln Glu Gly Thr Trp
450 455 460
Gln Trp Val Asp Gly Ser Pro Leu Leu Pro Ser Phe Lys Gln Tyr Trp
465 470 475 480
Asn Arg Gly Glu Pro Asn Asn Val Gly Glu Glu Asp Cys Ala Glu Phe
485 490 495
Ser Gly Asn Gly Trp Asn Asp Asp Lys Cys Asn Leu Ala Lys Phe Trp
500 505 510
Ile Cys Lys Lys Ser Ala Ala Ser Cys Ser Arg Asp Glu Glu Gln Phe
515 520 525
Leu Ser Pro Ala Pro Ala Thr Pro Asn Pro Pro Pro Ala
530 535 540
<210> 15
<211> 551
<212> PRT
<213> 人工序列
<400> 15
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr
20 25 30
Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His
35 40 45
Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe
50 55 60
Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg
65 70 75 80
Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys
85 90 95
Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu
100 105 110
Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His
115 120 125
Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly
130 135 140
Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln
145 150 155 160
Gly Glu Lys Thr Leu Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly
165 170 175
Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys
180 185 190
Ile Asp Ile Val Val Leu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
195 200 205
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly Glu Leu Ser Glu
225 230 235 240
Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Gln Leu Lys Ala
245 250 255
Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln
260 265 270
Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser
275 280 285
Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Trp Leu Lys Ala Ala Val
290 295 300
Gly Glu Leu Pro Glu Lys Ser Lys Met Gln Glu Ile Tyr Gln Glu Leu
305 310 315 320
Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gln
325 330 335
Gln Glu Ile Tyr Gln Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu
340 345 350
Leu Pro Glu Lys Ser Lys Gln Gln Glu Ile Tyr Gln Glu Leu Thr Arg
355 360 365
Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gln Gln Glu
370 375 380
Ile Tyr Gln Glu Leu Thr Gln Leu Lys Ala Ala Val Glu Arg Leu Cys
385 390 395 400
His Pro Cys Pro Trp Glu Trp Thr Phe Phe Gln Gly Asn Cys Tyr Phe
405 410 415
Met Ser Asn Ser Gln Arg Asn Trp His Asp Ser Ile Thr Ala Cys Lys
420 425 430
Glu Val Gly Ala Gln Leu Val Val Ile Lys Ser Ala Glu Glu Gln Asn
435 440 445
Phe Leu Gln Leu Gln Ser Ser Arg Ser Asn Arg Phe Thr Trp Met Gly
450 455 460
Leu Ser Asp Leu Asn Gln Glu Gly Thr Trp Gln Trp Val Asp Gly Ser
465 470 475 480
Pro Leu Leu Pro Ser Phe Lys Gln Tyr Trp Asn Arg Gly Glu Pro Asn
485 490 495
Asn Val Gly Glu Glu Asp Cys Ala Glu Phe Ser Gly Asn Gly Trp Asn
500 505 510
Asp Asp Lys Cys Asn Leu Ala Lys Phe Trp Ile Cys Lys Lys Ser Ala
515 520 525
Ala Ser Cys Ser Arg Asp Glu Glu Gln Phe Leu Ser Pro Ala Pro Ala
530 535 540
Thr Pro Asn Pro Pro Pro Ala
545 550
Claims (6)
1.一种在真核系统中表达CLD蛋白突变体的方法，其特征在于，制备的真核表达载体的重组蛋白CLD突变体的蛋白序列如SEQ ID NO.4所示。
2.一种如权利要求1所述方法制备得到的CLD蛋白突变体。
3.一种复合免疫原，包括如权利要求1所述方法制备得到的CLD蛋白突变体与HIV-1gp140。
4.权利要求2所述的CLD蛋白突变体或权利要求3所述的复合免疫原在制备抗HIV-1药物中的应用。
5.重组CLD蛋白与HIV-1gp140包膜蛋白组成的复合免疫原，所述的重组CLD蛋白为SEQID NO.12～SEQ ID NO.15中的任意一个蛋白。
6.权利要求5所述的复合免疫原在制备抗HIV-1药物中的应用。
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